5925
J . Am. Chem. SOC.1981, 103, 5925-5926 When the analytical oversight is corrected, a new meaning may be attached to the heat capacity function. Indeed, the meaning of the heat capacity function is contingent entirely upon how we formulate the equilibrium constant for the system. In this way the heat capacity function, either real or anomalous, provides a revealing mechanistic tool.
Nonequivalence of 31PNMR Chemical Shifts of RNA Complexes James L. Alderfer* and Geraldine L. Hazel Department of Biophysics, Roswell Park Memorial Institute Buffalo, New York 14263 Received March 17, 1981
c
J.-7 -------------1
-3
I
-4
-5
7 -
-6
Figure 1. Phosphorus-31 N M R spectra of (A) poly(A').poly(A+), (B) poly(A).poly(U), and (C) poly(A).2poly(U). Conditions are those inPhosphorus-3 1 nuclear magnetic resonance (3'P NMR) specdicated in Table I. troscopy has been used extensively in studies of nucleic acid constituents.'" Recently 31PN M R has been used to investigate helical complexes of larger oligonucleotides7and p~lynucleotides.~~~ Table I. P-31 Chemical Shiftsa of RNA Helical Complexes In the study of (dG-dC)J and poly(dG-dC): an important feature complexb chemical shift, ppm of the 3'P N M R spectrum of the helical complex is the conversion of a single resonance in low salt into two resonances in high salt. The high salt spectrum is consistent with the results of X-ray diffraction studies of oligomers of (dG-dC),'O which indicate two different phosphate-backbone conformations, one for ..GpC.. and another for ..CpG.. sequences (Z-DNA). Studies of the crystal a Maximum intensity position of resonance with respect t o instructure of d-pApTpApT" and a variety of existing physical and ternal trimethyl phosphate, 30 "C. Complex is 20-30 mM in chemical data on poly(dA-dT) lead to the hypothesis of an albase pair, in aqueous solution (20% D,O) containing 0.003 M ternating B-DNA structure'* for poly(dA-dT).poly(dA-dT).In EDTA, 0.04 M sodium phosphate and NaCl. The solution pHs are this model the phosphate backbone is different for ..APT.. and 6.8-7.2 except for poly(A+).poly(A+) (pH 5.6). Concentrations ..TpA.. sequences in the helical complex. This hypothesis has of NaCl are 0.05 M, poly(At).poly(At); 0.1 M, poly(A).poly(U); 0 and 0.6 M, poly(I).poly(C); 0.6 M, poly(I)~poly(C,,U); 0.4 M, received experimental support from recent 31PNMR studies9 of Chemical shifts of noncom lexed poly(A) poly(A).Zpoly(U). poly(dA-dT) where two phosphorus resonances are observed, and poly(U) are -3.97 and -3.85, respectively. BChemical shifts depending on the temperature and salt concentration. These data of noncomplexed poly(1) and poly(C) are -3.70 and -4.22, respecsupport the general contention that base sequence can affect tively. e Complexed phosphates of poly(1) strand. Complexed nucleic acid c~nformation.'~ phosphates of poly(C) strand. g Complexed phosphates of
--
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(1) Gorenstein, D. G. In "Nuclear Magnetic Resonance Spectroscopy in Molecular Biology"; Pullman, B., Ed.; D. Reidel: Dordrecht, Holland, 1978; pp 1-15. (2) (a) Patel, D. J. Biochemistry 1974, 13, 2388-239s. (b) Patel, D. J. Acc. Chem. Res. 1979, 12, 118-125. (3) (a) Reinhardt, C. G.; Krugh, T. R. Biochemistry 1977,16,2890-2895. (b) Krugh, T. R.; Nuss, M. E. "Biological Applications of Magnetic Resonance"; Academic Press: New York, 1979; pp 113--175. (4) (a) Akasaka, K.; Yamada, A,; Hatano, H. FEES Lett. 1975, 53, 339-341. (b) Yamada, A,; Kaneko, II.; Akasaka, K.; Hatano, H. Ibid. 1978, 93, 16-18. ( 5 ) (a) Klevan, L.;Armitage, I. M.; Crothers, D. M. Nucleic Acids Res. 1979, 6 , 1607-1616. (b) Davanloo, P.; Armitage, I. M.; Crothers, D. M. Biopolymers 1979,18,663-680. (c) Hogan, M. E.; Jardetzky, 0.Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 6341-6345. (d) Bolton, P. H.; James, T. L. J . Am. Chem. SOC.1980, 102 25-31. (e) Shindo, H. Biopolymers 1980, 19, 509-522. ( 6 ) (a) Mariam, Y. H.; Wilson, W. D. Biochem. Biophys. Res. Commun. 1979,88,861-866. (b) Jones, R. L,.;Wilson, W. D. J . Am. Chem. SOC.1980, 102, 7778-7779. (7) Patel, D. J.; Canuel, L. L.; Pohl, F. M. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 2508-2511. (8) SimDson. R. T.; Shindo. H. Nucleic Acrds Res. 1980, 8, 2093-2103. (9) (a) Simpson, R. T.; Shindo, H. Nucleic Acids Res. 1979, 7, 481-492. (b) Shindo, H.; Simpson, R. T.; Cohen, J. S. J . Bioi. Chem. 1979, 254, 8125-8128. (10) (a) Wang, A. H.-J.; Quigley, G. J.; Kolpak, F. J.; Crawford, J. L.; van Boom, J. H.; van der Marel, G.; Rich, A. Nature (London) 1979, 282, 680-686. (b) Drew, H.; Takano, T.; Tanako, S., Itakura, K.; Dickerson, R. E. Ibid. 1980, 286, 587-573. (c) Wang, A. H . J . ; Quigley, G. J.; Kolpak, F. J.; van der Marel, G.; van Boom, J. H.; Rich, A. Science (Washington) 1981, 211, 171-176. (11) Viswamitra, M. A.; Kennard, 0.;Jones, P. G.; Sheldrick, G. M.; Salisburg, S.; Falvello, L.; Shakked, Z . Nature (London) 1978, 273,687-688. (12) Klug, A,; Jack, A,; Viswamitra, M. A,; Kennard, 0.;Shakked, Z.; Steitz, T. A. J . Mol. Biol. 1979, 131, 669-680. (13) Wells, R. D.; Blakesley, R. W.; Hardres, S. C.; Horn, G. T.; Carson, J. E.; Selsing, E.; Burd, J. F.; Chan, H. W.; Dodgson, J. B.; Jensen, K. F.; Nes, I. F.; Wartell, R. M . CRC Crit. Reo. Biochem. 1977, 5, 305-340.
poly(U) strand involved in Hoogsteiii base pair with poly(A).
Variation in the structure of the phosphate backbone is also apparent in fibers of nucleic acid^.'^^'^ Fibers of RNA homopolynucleotide complexes have been extensively studied by X-ray diffraction to deduce their helical conformations. From these studies it is apparent that phosphate-backbone conformations may be dependent not only on the base sequence but also on the nature of the association of bases through hydrogen bonds in polynucleotide complexes. In the cases of complexes with poly(A), three types of base-base association are observed: poly(A+). poly( A'), poly( A).poly( U) , I 7 and poly( A).2poly( U) .I * The general feature of the phosphate-backbone conformation of the poly(A+).poly(A+) and poly(A)+poly(U)complexes is that of C, symmetry with respect to the helical axis of the double-stranded complex (Le., the conformation of the phosphate backbone of each strand is identical). In contrast, the fiber structure of the poly(A).Zpoly(U) complex is deduced as one in which a unique phosphate-backbone conformation exists for each of the three strands.
'
(14) (a) Bram, S.; Tougard, P. Nature (London) 1972,239, 128-131. (b) Bram, S. Biochem. Biophys. Res. Commun. 1972, 48, 1088-1092. (15) Arnott, S.; Chandrasekaran, R.; Selsing, E. In "Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions" Sandaralingam, M., Rao, S. T., Eds.; University Park Press: Baltimore, 1975; pp 577-596. (16) (a) Rich, A.; Davies, D. R.; Crick, F. H. C.; Watson, J. D. J . Mol. Biol. 1961, 3, 71-86. (b) Holcomb, D. N.; Tinoco, I., Jr. Biopolymers 1965, 3, 121-133. (17) Arnott, S.; Hukins, D. W. L.; Dover, S. D.; Fuller, W.; Hodgson, A. R. J . Mol. Biol. 1973, 81, 107-122. (18) (a) Arnott, S.; Bond, P. J. Nature (London) 1973,244,99-101. (b) Miles, H. T.; Frazier, J. Biochem. Biophys. Res. Commun. 1964, 14, 21-28.
0002-7863/8 1/ 1503-5925$01.25/0 0 1981 American Chemical Society
5926
J. Am. Chem. SOC.1981,103, 5926-5928
We have studied these three polyribonucleotide complexes19 in aqueous solution by 31PN M R spectroscopy (80.6 MHz) above (spectra not shown) and below the thermal denaturation temperature (T,) (Figure 1) of the helical complex. Above the T,, the spectral lines are generally sharp (Avl12 C 2 Hz) and consist of one or two resonances of expected intensity,22 reflecting the homopolymer stoichiometry of the respective complexes. Below the T, (Figure l ) , the resonances are somewhat broader ( A v I l 2 20-50 Hz),presumably reflecting effects due to reduced correlation times of the phosphates of the base-paired complexes. Additional features of the spectra of these complexes are as follows: (a) poly(A+)-poly(A+) has one phosphorus resonance;23 (b) poly(A).poly(U) has two resonances of approximately equal intensity; (c) poly(A).2poly(U) has three resonances, each of approximately equal intensity. The chemical shift positions of the resonances of these and additional polynucleotide complexes are listed in Table I. An unanticipated feature of these spectra is the observation of multiple resonances in three of the doublestranded helical complexes [poly(A).poly(U), poly(1)-poly(C),and poly(I)-poly(C12U)].25 Plausible explanations for the observation of more than one resonance are (1) two equal populations of helical complexes having different phosphate-backbone conformations, each having C2symmetry with an exchange rate slow on the NMR time scale, (2) a single helical complex having C2 symmetry but containing local phosphate magnetic anisotropy2’ (e.g., due to solution environment30), and (3) a single helical complex where the two strands have different phosphate-backbone conformations. Various studies of these complexes (effects of temperature, ionic strength, RNA-DNA hybrid complexes) do not support the first e ~ p l a n a t i o nand ~ ~ provide tentative assignments” of some of the
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(19) Polynucleotide complexes were prepared as previously described.20 These complexes were sonicated by using a Branson Sonifier (Model W185; equipped with a tapered micro tip) at 0 OC under nitrogen atmosphere. Integrity of the complex was determined by using thermal denaturation profile (UV spectroscopy) before and after sonication, and after sonication the complex had a broad molecular weight distribution as determined from polyacrylamide gel electrophoresis (7.5%) in 40 mM Tris acetate (pH 8.0). The size was generally greater than 150 base pairs, using the Hae 111 restriction fragments of PM2 DNA as comparative size markers.21 (20) Carter, W. A.; Pitha, P. M.; Marshall, L. W.; Tazawa, I.; Tazawa, S.; Ts’o, P. 0. P. J . Mol. Biol. 1972, 70, 567-587. (21) Kovacie, R. T.; van Holde, K. E. Biochemistry 1977, 16, 149G1498. (22) Spectra are acquired by using conditions where accurate resonance intensities can be obtained. An inverted-gated decoupling (proton decoupling without NOE enhancement) acquisition scheme is used on a WP-200 NMR spectrometer. Pulse repetition rates of 2 1 0 s and flip angles of 536’ are utilized. (23) A similar observation has recently been rep~rted.~‘ (24) Lerner, D. B.; Kearns, D. R. Biopolymers 1981, 20, 803-806. (25) In the alternating copolymer poly(1-C), two phosphorus resonances have been observedz6(-4.48 and -4.25 ppm, 35.5 “C). The difference in these resonances (A6 0.23) is about one-half that observed (Table I) for poly(1). poly(C) (A6 = 0.51). The smaller A6 for poly(1-C) does not appear due to line-width differences since they are not symmetrically positioned between the resonances of poly(I).poly(C). The two resonances in poly(1-C) may reflect sequence variations in the sugar-phosphate-backbone conformation. (26) Patel, D. J. In “Nucleic Acid Geometry and Dynamics”; Sarma, R. H., Ed.; Pergamon Press: New York, 1980; pp 185-231. (27) A reasonable contributor here might be ring-current anisotropy from different base moieties on the phosphates of the individual strands. Ringcurrent calculations have been made for homopolymers in the A-RNA conformation2*by using the equivalent dipole modeLZ9 The chemical shift difference (A6) of the phosphates of different strands is 0.09 ppm for poly(A).poly(U) and 0.05 ppm for poly(I).poly(C). While it is apparent ringcurrent anisotropy may contribute to chemical shift differences, this calculated effect is not sufficient to account for the observed results. (28) Amott, S.; Hukins, D. W. L. Biochem. Biophys. Res. Commun. 1972, 48, 1392-1399. (29) Abraham, R. J. in “Nuclear Magnetic Resonance Spectroscopy in Molecular Biology”; Pullman, B., Ed.; D. Reidel: Dordrecht, Holland, 1978; pp 461-479. (30) Solutesolvent interactions” or specific interactions mediated by the base moiety (e&, guanine.Mgz+.H20.phosphate’h) could generate anisotropy. (31) Lerner, D. B.; Kearns, D. R. J . Am. Chem. Sor. 1980, 102, 7611-7612. (32) In all cases (Table I) where multiple resonances are observed, their intensities disappear at essentially the same rate on going through the thermal denaturation temperature and reappear at the same rate on renaturation; this is also observed for poly(I).poly(C) and poly(I).poly(C12U) at NaCl concentrations between 0-0.6 M and for poly(A).poly(U) at low salt concentrations.
0002-7863/81/l503-5926$01.25/0
base-paired resonances in Table I. The second and third explanations are both considered possible contributors to the multiple resonances observed. Our collective results from these complexes in various salts35 of poly(A).Spoly(U) at conditions where the triple-to-double-stranded conversion occurs, and the observed three resonance pattern of poly(A).2poly(U) anticipated from the unique strand conformations of the fiber,lSa strongly suggest that the third explanation (strands with different phosphate-backbone conformations) is a reasonable contributor to the multiple resonances observed in these complexes. Additional experiments are necessary to unequivocally assign the resonances to specific strands and to determine the relative contributions of mechanisms 2 and 3 to the observed chemical shifts. However, irrespective of the explanation of the resonance multiplicity, these results illustrate the utility of the 31PN M R technique for probing the phosphate backbone of individual strands in helical complexes. Acknowledgment. We acknowledge the National Science Foundation and National Institutes of Health (CA-25438) for support in purchasing the N M R spectrometer and Franklyn Wissler for performing the ring-current calculations. (33) (a) The poly(I)~poly(C)helical resonances are assigned by comparison to poly(dI).poly(C) (-4.27 and -4.66 ppm) by assuming the two resonances most similar in chemical shift are those from the RNA strand. (b) In poly(A).2poly(U), the Hoogstein base-paired poly(U) strand (lowest field resonance) was assigned by comparison of the poly(A).poly(U) and poly(A). 2poly(U) spectra and by a temperature study of poly(A).2poly(U) at low salt where the 3 2 1 stranded conversion” occurs. At this condition the lowest field resonance disappeared and reappeared as a sharp line with a chemical shift expected for free poly(U), while the intensity of the other two higher-field resonances remained constant. (34) Stevens, C. L.; Felsenfeld, G. Biopolymers 1964, 2, 293-314. (35) In the case of poly(I).poly(C), similar results are obtained in the presence or absence of added NaCl or in the presence of added MgCI2.
-+
Reaction between HOy and Chlorine in Aqueous Solution E. Bjergbakke R i m National Laboratory DK-4000, Roskilde, Denmark
S. Navaratnam and B. J. Parsons School of Natural Sciences, Kelsterton College North E . Wales Institute, Connah’s Quay Clwyd, CH5 4BR, United Kingdom A. J. Swallow* Paterson Laboratories, Christie Hospital and Holt Radium Institute Manchester M20 9BX, United Kingdom Received May 1 , 1981
Reactions in aqueous solution containing ferrous sulfate are among the best u n d e r s t d of radiolytic reaction mechanisms, but hitherto unsuspected reactions must be taking place when large radiation doses are applied instantaneously in the presence of added chloride.’ We have recently found indications that yields under these conditions might be explicable if H02. radicals reduce C12.2 This communication reports independent confirmation of this reaction, obtained in a system designed to demonstrate it. (1) Navaratnam, S.; Parsons, B. J.; Swallow, A. J. Radiat. Phys. Chem. 1980, 15, 159. (2) Bjergbakke, E.; Navaratnam, S.;Parsons, B. .I.; Swallow, A. J., manuscript in preparation. (3) Gilbert, C. W.; Ingalls, R. B.; Swallow, A. J. Radial. Phys. Chem. 1977, 10, 221. (4) Jayson, G. G.; Parsons, B. J.; Swallow, A. J. J . Chem. Sot., Faraday Trans. 1 1973, 69, 1597. ( 5 ) Swallow, A. J. “Radiation Chemistry”; Longman: London, 1973. (6) Hasegawa, K.; Neta, P. J . Phys. Chem. 1978, 82, 854. (7) Scott, R. L. J . Am. Chem. SOC.1953, 75, 1550.
0 1981 American Chemical Society
